A fijiviral nonstructural protein triggers cell death in plant and bacterial cells via its transmembrane domain

Abstract Southern rice black‐streaked dwarf virus (SRBSDV; Fijivirus, Reoviridae) has become a threat to cereal production in East Asia in recent years. Our previous cytopathologic studies have suggested that SRBSDV induces a process resembling programmed cell death in infected tissues that results in distinctive growth abnormalities. The viral product responsible for the cell death, however, remains unknown. Here P9‐2 protein, but not its RNA, was shown to induce cell death in Escherichia coli and plant cells when expressed either locally with a transient expression vector or systemically using a heterologous virus. Both computer prediction and fluorescent assays indicated that the viral nonstructural protein was targeted to the plasma membrane (PM) and further modification of its subcellular localization abolished its ability to induce cell death, indicating that its PM localization was required for the cell death induction. P9‐2 was predicted to harbour two transmembrane helices within its central hydrophobic domain. A series of mutation assays further showed that its central transmembrane hydrophobic domain was crucial for cell death induction and that its conserved F90, Y101, and L103 amino acid residues could play synergistic roles in maintaining its ability to induce cell death. Its homologues in other fijiviruses also induced cell death in plant and bacterial cells, implying that the fijiviral nonstructural protein may trigger cell death by targeting conserved cellular factors or via a highly conserved mechanism.

. Within the tumour phloem, SEs, cells that are terminally enucleated and differentiated, show unexpected hyperplasia together with PPs and aggregate into a special region lacking companion cells (CCs), which is markedly different from the staggered pattern of SEs and CCs in healthy phloem , suggesting a process similar to (programmed) cell death in the infected tissues. However, it is not known which viral product is responsible for this cell death.
Southern rice black-streaked dwarf virus (SRBSDV) (Zhou et al., 2008), also known as Rice black-streaked dwarf virus 2 (RBSDV-2) , is a newly recognized member of genus Fijivirus (Adams et al., 2016). The virus is a destructive pathogen that has been threatening the production of rice, maize, and other gramineous crop plants in East Asia in recent years . Its virion is icosahedral but appears spherical in shape and consists of two concentric layers of capsid protein with an overall diameter of 75-85 nm, surrounding 10 linear double-stranded (ds) RNA genome segments (Hoang et al., 2011). In the field, the virus is transmitted by the white-backed planthopper (WBPH, Sogatella furcifera), a migratory pest, in a persistentpropagative manner and the viral disease has thus rapidly expanded among rice-growing areas of Vietnam, China, and Japan in recent years.
In China alone, its outbreaks in 2010 affected more than 1,360,000 ha of rice-growing areas, resulting in 30%-50% yield losses and even no harvest in some seriously infected rice paddies . The viral 10 linear dsRNA genomic segments are named S1-S10 according to their migration from slow to fast in polyacrylamide gel electrophoresis (PAGE) (Wang et al., 2010). Genomic sequence analysis shows that S1-S4, S6, S8, and S10 are monocistronic, encoding proteins P1-P4, P6, P8, and P10, respectively, but S5, S7, and S9 each contain two open reading frames (ORFs). The proteins encoded by the upstream ORFs are called P5-1, P7-1, and P9-1, whereas those encoded by the downstream ORFs are called P5-2, P7-2, and P9-2. The entire genome of SRBSDV thus has at least 13 viral genes, of which six, P1-P4, P8, and P10, encode structural proteins that are assembled into the virions Wang et al., 2010). Biochemical and immunological experiments indicate that P5-1, P6, and P9-1 are involved in the formation of viroplasms (Li et al., 2013(Li et al., , 2015, a type of discrete cytoplasmic inclusion body that is a factory for virus replication and assembly in infected host plants  and vector planthoppers (Jia et al., 2012;Mao et al., 2013). The nonstructural protein P7-1 is a viral component of the tubular structures that are typical in reoviral infections (Liu et al., 2011) and which are thought to play key roles in the intercellular movement of virions within insects (Jia et al., 2014). The other proteins of SRBSDV also seem to be similar to their counterparts in other fijiviruses in size and amino acid sequences, and may be involved in virus-host or virus-vector interactions; however, their functions remain unknown.
In host plants, SRBSDV induces some typical symptoms, including dwarfing and swellings or tumours, which are often found along the leaf sheath or on the underside of the leaf blade, and along the veins on stem of rice (Wu et al., 2013) and maize (Hoang et al., 2011). These swellings or tumours are derived from the hyperplasia of phloem tissues but are obviously different from healthy phloem tissues in cell types and frequency of intercellular gateways, which may provide a better microenvironment for SRBSDV multiplication and movement . In the SRBSDV-induced tumours, SE hyperplasia and the special region composed exclusively of SE are also observed. As the process of SE differentiation resembles an arrested cell death (Furuta et al., 2014;Geldner, 2014), we suggested that SRBSDV might encode a protein pronouncedly affecting cell viability. To test this hypothesis, we have now investigated the ability of each SRBSDV-encoded protein to induce cell death in plant cells and/or in the bacterium Escherichia coli.

| Screening of SRBSDV-encoded proteins that affect plant cell viability
To determine whether protein(s) encoded by SRBSDV have the ability to induce cell death, the 13 SRBSDV-encoded proteins were individually expressed by Agrobacterium tumefaciens-mediated transient expression, a procedure that has been widely used to detect the cytopathogenic effects of viral (Qian et al., 2016) or nonviral effector proteins (Franco-Orozco et al., 2017;Yu et al., 2012). Briefly, each SRBSDV protein was expressed under the control of double cauliflower mosaic virus (CaMV) 35S promoters in Nicotiana benthamiana leaves through agroinfiltration. The jellyfish green fluorescent protein (GFP) was used as a negative control, with XEG1, a Phytophthora sojae effector protein that causes cell death, used as a positive control in plants (Ma et al., 2015). As expected, necrosis was observed 3 days after agroinfiltration in leaf patches expressing XEG1 but not in patches expressing GFP ( Figure 1a).
Interestingly, SRBSDV P9-2, a nonstructural protein of unknown function, was also observed to induce necrosis similar to, although less pronounced than, that caused by XEG1 ( Figure 1a). All other SRBSDV proteins behaved like GFP in agroinfiltrated leaf patches ( Figure 1a). Trypan blue staining, an assay for the visualization of cell death (Cooksey, 2014;Keogh et al., 1980), further confirmed the occurrence of cell death in the leaf patches agroinfiltrated with the 35S:P9-2 construct (Figure 1b) as in those expressing XEG1, clearly indicating that P9-2 could induce cell death in plant cells.
The burst of reactive oxygen species (ROS) is thought to be a hallmark of cell death (Breusegem & Dat, 2006;Fulda, 2016).
Consistent with this, leaf patches expressing XEG1 or P9-2, but not those expressing GFP or other SRBSDV proteins, could be readily stained by 3,3′-diaminobenzidine (DAB), a dye that has been widely used to detect H 2 O 2 ( Figure 1b) (Thordal-Christensen et al., 1997), demonstrating that P9-2 also induced cell death by a burst of ROS.

| P9-2 induces systemic symptoms with necrosis when expressed by a heterologous virus
To investigate whether P9-2 induces plant cell death when expressed by a different virus, the P9-2 ORF was cloned into the tobacco rattle virus (TRV) vector pTRV2 (Liu et al., 2002). Agrobacterium cultures harbouring this recombinant plasmid were infiltrated into N. benthamiana leaves together with those harbouring pTRV1. Like the tran- TRV has been widely used as a tool to silence endogenous genes (Liu et al., 2002). To rule out the possibility that the cell death induced by TRV-P9-2 might be caused by silencing of an unknown plant gene, TRV carrying the P9-2 ORF but without a start codon (TRV-ΔP9-2) was constructed.
N. benthamiana inoculated with this construct showed no difference to those inoculated with TRV-GFP in both inoculated and systemic leaves ( Figure S1). Thus, even if the small interfering RNAs derived from the P9-2 ORF targeted some endogenous genes, this did not lead to cell death, supporting the view that cell death is induced by P9-2 at the level of protein rather than RNA.

| P9-2 induces cell death in E. coli
In attempts to prepare an antiserum against P9-2, we failed to express the protein in prokaryotic expression systems. This prompted us to test the idea that P9-2 might also induce cell death in prokaryotic cells. The P9-2 ORF was therefore subcloned into the prokaryotic expression vector pET32a and the resulting plasmid, pET32a-P9-2, was transformed into E. coli BL21 (DE3) pLysS. pET32a-GFP was obtained similarly and used as a negative control. In the absence of isopropylβ-D-thiogalactoside (IPTG), an inducer for prokaryotic expression, the growth of the E. coli carrying pET32a-P9-2 was comparable to, although slightly slower than, those carrying pET32a-GFP ( Figure 3a).
IPTG had negligible effects on the E. coli carrying pET32a-GFP.
However, it seriously inhibited the growth of E. coli carrying pET32a-P9-2 ( Figure 3a). Aliquots of each culture were pipetted out 3.5 h after IPTG addition, plated on solid Luria-Bertani (LB) medium after a 1000fold dilution and allowed to grow overnight. As shown in Figure 3b, numerous colonies were found on the medium plated with the E. coli carrying pET32a-GFP, irrespective of IPTG addition, and also from the uninduced E. coli carrying pET32a-P9-2. However, only a small number of colonies were observed on the medium plated with the IPTGinduced E. coli carrying pET32a-P9-2.
As an independent approach, 4-methylumbelliferylβ-D-glucuro nic acid (MUG) was added to overnight cell cultures of the E. coli carrying pET32a-P9-2/pET32a-GFP, alone or together with IPTG. Three hours after the addition, the E. coli was observed under UV light.
Because living E. coli can hydrolyse MUG to 4-methylumbelliferone (4-MU), which emits blue light under UV light excitation, the intensity of the blue fluorescence was an indicator of the amounts of living E. coli cells. As shown in Figure 3c, IPTG had no visible effect on the fluorescence intensity of E. coli carrying pET32a-GFP, but it greatly reduced the fluorescence intensity of the E. coli carrying pET32a-P9-2, showing that the expression of P9-2 also could destroy the viability of prokaryotic cells.

| Plasma membrane localization of P9-2 is required for cell death induction
Subcellular localization of a protein may provide a clue to its functions and we therefore predicted the subcellular localization of P9-2 using two independent web-based programs that make predictions based on the protein amino acid sequences: PSORT v. 6.4, (https://psort. hgc.jp/) and YLoc-HighRes (https://abi-servi ces.infor matik.uni-tuebi ngen.de/yloc/webloc.cgi). Both packages indicated that P9-2 might most probably (60%-80%) be targeted to plasma membranes. To validate its subcellular localization, binary vectors expressing P9-2 fused at its N-or C-terminus with the GFP and Arabidopsis plasma membrane intrinsic protein 2A (AtPIP2A), a marker labelling plasma membrane in plant cells (Nelson et al., 2007), fused with m-Cherry, were constructed and coinfiltrated into N. benthamiana epidermal cells using the Agrobacterium-mediated method. To decrease the F I G U R E 2 Symptoms on the inoculated leaves (a), petioles (b), stems (c), and upper leaves (d) of Nicotiana benthamiana plants inoculated with TRV expressiing the SRBSDV protein P9-2. TRV expressing GFP was used as a negative control. dpi, days postinoculation.
The growth curves of E. coli BL21 (DE3) pLysS containing pET32a-GFP or pET32a-p9-2 grown in Luria-Bertani (LB) broth with or without isopropylβ-D-thiogalactoside (IPTG) induction. Cell growth was monitored by OD 600 . (b) E. coli BL21 (DE3) pLysS containing pET32a-GFP (I and II) or pET32a-p9-2 (III and IV) were growth on an LB plate for 16 h with (II and III) or without IPTG (I and IV). (c) 4-methylumbelliferylβ-D-glucuronic acid (MUG) was added to overnight cell cultures of the E. coli BL21 (DE3) pLysS containing pET32a-p9-2/pET32a-GFP, alone or together with IPTG. At 3 h after the addition, the cultures of E. coli BL21 (DE3) pLysS growth were monitored under UV light. Each experiment was performed at least three times. effect of cell death on the subcellular localization, the fluorescence was monitored by confocal microscopy 30 h after infiltration, an early stage of cell death. In the coinfiltrated cells, the fluorescence from fused P9-2/GFP was mainly distributed on plasma membranes and most of the GFP fluorescence was colocalized with that from fused AtPIP2A/mCherry (Figure 4a). In a subcellular fractionation assay, P9-2 was mainly detected in the plasma membrane compartment ( Figure S2), further supporting its subcellular localization on the plasma membrane. Interestingly, when its N-terminus was fused with a sequence for the nuclear localization signal (NLS) from SV40 (Kalderon et al., 1984), the modified P9-2 protein was trans- were observed when P9-2 was artificially relocalized into the endoplasmic reticulum (ER) lumen or bound onto the F-actin cytoskeleton by attaching an ER signal peptide/retention signal (HDEL) (Gomord et al., 1997;Nelson et al., 2007) or Lifeact, a 17 amino acid peptide used as a versatile marker to visualize F-actin (Lv et al., 2017c;Riedl et al., 2008). Taken together, these results indicate that the plasma membrane localization of P9-2 protein could be required for its ability to induce cell death.

| Transmembrane helices of P9-2 were crucial for cell death induction
To further investigate its functional domains, two transmembrane helices of P9-2 were identified spanning amino acid residues 80-104 and 116-140 using the transmembrane prediction servers, Phobius and Nilaparvata lugens reovirus (NLRV) (Nakashima et al., 1996). were substituted with aspartic acid (D), a polar amino acid residue with negative charge, were constructed and tested as above. The mutant P9-2-F90D appeared to induce much less cell death than any other single site-directed mutant while the effects of P9-2-L103D were similar to those of P9-2-L103A and intact P9-2, supporting the importance of F90. Because three of the four conserved sites were located within the first transmembrane helix, we hypothesized that the first transmembrane helix may be more important for cell death induction and that the conserved amino acids may have synergistic effects so that a single amino acid mutation might not be enough to abolish its function.
Based on this idea, three double mutants of SRBSDV P9-2 were created. All the three mutants were each expressed with the TRV vector ( Figure S6) and they still appeared to induce cell death, although to varying degrees (Figure 6b). Among above mutants, the double mutant Although further studies are needed to explain these observations, altogether they support the notion that the first transmembrane helix within the central hydrophobic region could be crucial for cell death induction and that the conserved F90, Y101, and L103 amino acid residues could play synergistic roles in maintaining the ability of P9-2 to induce cell death.

| P9-2 activity is conserved in fijiviruses
SRBSDV P9-2 and its homologues are conserved among fijiviruses, sharing 21%-73% amino acid identity Xue et al., 2014). However, these proteins do not exhibit any significant sequence similarity to other reoviral (or indeed any other viral) protein, suggesting that P9-2 may be unique to fijiviruses. Further multiple alignment suggested that a central hydrophobic region was always present in fijiviral P9-2 homologues and two transmembrane helices could be predicted using the transmembrane topology prediction servers, Phobius (https://phobi us.sbc.su.se/) and TMHMM v. 2.0 (http://www.cbs.dtu.dk/servi ces/TMHMM/) ( Figure S5). The similarity suggests that the ability of P9-2 homologues to induce cell death may be conserved among fijiviruses. To confirm this speculation, we also cloned the full-length coding region of P9-2 from Maize rough dwarf virus (MRDV), another member of the genus Fijivirus (Lv et al., 2016), and tested its cytopathogenic effects in a similar way.
As expected, MRDV P9-2 also induced cell death either when ex- our previous studies to prepare antisera, we also failed to express the full-length P9-2 protein of RBSDV and SRBSDV due to poor cell survival rate. However, in these three independent studies, deletion of the central hydrophobic domain resulted in successful expression of the proteins from different fijiviruses, also suggesting that the central hydrophobic domains play an important role in the cell death induction. Thus, the cell-death induction activity and the mechanisms underlying this activity may be universal for P9-2 among all fijiviruses.

| DISCUSS ION
Our previous studies suggest that a product of SRBSDV may induce the SE differentiation by cell death-like or a near-death process, but an experimental system allowing us to identify the effect of a viral protein on SE differentiation is unavailable so far. However, because SE differentiation resembles a specific form of cell death (Furuta et al., 2014), the results from this study are at the least  Guo et al., 2019;Botrytis cinerea transglycosylase, Bi et al., 2021).
Generally, one of the following two mechanisms is involved. First, the protein can be recognized by a resistance ( R) protein from the plant, either directly or indirectly. The R protein then triggers a signal transduction cascade, leading to rapid cell death (Coll et al., 2011;de Ronde et al., 2014;Sun et al., 2020). This type of cell death, commonly known as a hypersensitive reaction, is a defence mechanism used by plants to combat infections (Kombrink & Schmelzer, 2001;Park, 2005). Second, the protein may activate or hijack a prodeath signal cascade. In this case, the cell death induced by the protein can be blocked by silencing one or several host genes (Yu et al., 2012;Zhang et al., 2020). In N. benthamiana plants, P9-2 appeared to activate the expression of marker genes for programmed cell death (PCD) (Figure S7), supporting that the viral protein could induce a process resembling PCD in plants. Because N. benthamiana is not a host of SRBSDV or any related fijiviruses, it seems unlikely that this plant has an R protein to specifically recognize P9-2. Therefore, we prefer the second possibility for the cell death induction of P9-2.
Our results showed that P9-2 also induces cell death in E. coli, a model prokaryotic cell. The hydrophobic domain necessary for cell death induction in plants is also important for cell death induction in E. coli, suggesting that the same mechanism is used by P9-2 to induce cell death in the two very different systems. This seems to be a very in mammalian cells undergoing PCD, pointing to a conserved mechanism. Thus, it is possible that P9-2 may activate a prodeath pathway that is conserved among prokaryotes and eukaryotes. In this regard, it is worth noting that HAP contains two transmembrane domains like P9-2, although the role of the transmembrane domains in the action of HAP has yet to be determined (Gan et al., 2004).
All fijiviruses encode P9-2 or a homologue as the downstream ORF within corresponding genomic positions (FDV, Soo et al., 1998;RBSDV, Isogai et al., 1998a;MRCV, Guzmán et al., 2007;MRDV, Xie et al., 2017b;SRBSDV, Xue et al., 2014). Our previous studies showed that no subgenomic RNAs for downstream ORFs can be detected in fijivirus-infected plants and that there are no known internal ribosome entry sites to express these 3′-proximal ORFs of bicistronic dsRNAs (Lv et al., 2012;Yang et al., 2014). The only plausible mechanism by which these downstream ORFs can be expressed is restarting or leaky scanning (Li et al., 2011), which is sup- fects of P9-2 on SE differentiation might best be conducted using lower expression levels. However, the findings of this study may still have biological significance for at least the following two putative reasons: (i) P9-2 may induce the death of certain cell type (i.e., companion cell) at low concentrations, as predicted from the idea that it may function through triggering a signal cascade, which might lead to lack of companion cells within SRBSDV-induced tumours . (ii) P9-2 may induce a very mild form of cell death or near-death differentiation when expressed at low concentrations, which might regulate SE specification and differentiation from the division of phloem-parenchyma cells, leading to SE hyperplasia and the de novo formation of an SE-SE specific region within SRBSDVinduced tumours . Although the information on the ontogeny of SRBSDV-induced SE-SE specific region and the role of P9-2 in SE differentiation remains very limited, this is therefore the first step to dissect a viral factor that may be involved in SE differentiation, which may link SE differentiation to a mild form of cell death.

| Plant materials
All N. benthamiana plants were grown in a growth chamber at 25°C with 16 h light/8 h dark and 70% relative humidity as previously described. Infected leaves were collected from diseased rice plants in Zhejiang Province and stored at −80°C until use.

| Plasmid construction
A pCAMBIA L1300-derived binary vector ( Figure S8) was used for transient expression. The ORFs specifying GFP, XEG1 or SRBSDV proteins were each amplified from GFP-or XEG1-containing plasmids or SRBSDV genomic cDNAs by PCR with primer pairs (Table S1). The PCR products were recombined into the pDONR201 Gateway vector with BP clonase (Invitrogen) and sequenced, before inserting into the binary vector with LR clonase (Invitrogen).
The TRV-based vector ( Figure S9) was used to express GFP, XEG1, P9-2 or P9-2 mutants in N. benthamiana. To do this, each fragment of interest was amplified by conventional or overlapping PCR with primer pairs (Table S2) and the PCR product was inserted into the ClaI/SalI site of the vector pTRV2 using a ClonExpress II One Step Cloning Kit (Vazyme). pET32a (Novagen) was used to express GFP, P9-2 or P9-2 mutants in E. coli. To do this, each fragment of interest was amplified by PCR with primer pairs (Table S3) and the PCR product was inserted into the EcoRI/XhoI site of the vector pET32a.
For subcellular localization studies, the expression cassettes of P9-2/GFP and AtPIP2A/mCherry fusions were inserted in a modified binary vector from pCAMBIA1300 ( Figure S8). To change the subcellular localization, the N-or C-ends of P9-2 were fused with the nuclear localization signal (NLS), endoplasmic reticulum (ER) signal peptide/retention signal (HDEL), or Lifeact sequences. The primer pairs for plasmid construction are listed in Table S4 and their schematic diagrams are shown in Figure S8.

| Agroinfiltration
Each recombinant binary construct described above was transformed

| DAB and trypan blue staining
DAB staining was performed according to a procedure described by Thordal-Christensen et al. (1997). Briefly, leaves were excised from N. benthamiana using a sterilized razor blade at 72 h after agroinfiltration. After incubation for 8 h in a solution of 1 mg/ml DAB-HCl (pH 3.8) at room temperature, the leaves were boiled in ethanol for 10 min and rinsed twice in double distilled water before photographing. Trypan blue staining was performed according to a procedure described by Keogh et al. (1980) and Koch and Slusarenko (1990).
Briefly, leaves were excised from N. benthamiana 72 h after agroinfiltration. After washing with double distilled water, the leaves were immersed and boiled in a trypan blue staining solution (10 ml lactic acid, 10 ml glycerol, 10 g phenol, 10 mg trypan blue, dissolved in 10 ml double distilled water) for 3 min, cooled at room temperature for 1 h, decoloured in 2.5 g/ml chloral hydrate for 48 h, and photographed.

| Western blotting
Agroinfiltrated leaves were harvested and ground in liquid nitrogen.
The powder (about 100 mg) was mixed with 100 μl of 2 × SDS loading buffer. After boiling for 5 min, the extracts were centrifuged at 12,000 × g for 5 min. The supernatant was loaded to and separated by 12.5% SDS-PAGE. Separated proteins were transferred to a nitrocellulose membrane and were probed with a commercially available antibody to the FLAG epitope (TransGen). A goat antirabbit immunoglobulin G (IgG) conjugated with alkaline phosphatase (Sigma-Aldrich) was used as the secondary antibody and the band was visualized by incubating the membranes in NBT-BCIP solution following the protocol from the manufacturer.

| Subcellular fractionation
An assay of cytoplasmic, nuclear, and membrane fractionation was performed using the Plant Nuclei and Cytoplasmic Protein Extraction Kit (BestBio) and Minute Plasma Membrane Protein Isolation kit (Invent) according to the manufacturer's protocols, respectively. The isolated proteins were subjected to SDS-PAGE and immunoblotting as described above. The primary antibodies against H + ATPase (Agrisera), UDP-glucose pyrophosphorylase (UGPase) (Agrisera), and histone H3 (Abclonal), were used as internal cellular compartment markers for plasma membrane, cytoplasm, and nucleus, respectively.

| The effects of P9-2 on E. coli
To obtain growth curves of E. coli expressing GFP, P9-2 or P9-2 mutants, each strain of E. coli was cultured in LB liquid medium supplemented with ampicillin at 37°C. An aliquot of the culture was inoculated to 10 ml of fresh LB and cultured to an OD 600 of about 0.5. An aliquot of the second culture was inoculated to 40 ml of fresh LB. The liquid, with an OD 600 of about 0.03, was cultured at 37°C with shaking. About 100 μl of the culture was pipetted out every 1 h for OD 600 measurement. When the value of OD 600 reached 0.5, IPTG was added to the culture at a final concentration of 0.4 μM.
OD 600 was measured every 0.5 h after IPTG addition.
To confirm cell death with MUG, a procedure described by Feng and Hartman (1982) was employed. Briefly, the E. coli BL21 (DE3) pLysS carrying each recombinant construct was cultured in LB liquid medium supplemented with ampicillin. An aliquot of the culture was inoculated to 10 ml of fresh LB and cultured to an OD 600 of 0.5-0.8. The cells were harvested by a centrifuge at 3000 × g for 3 min and the pellet was resuspended with 2 ml of LB. The 2 ml of suspension was divided into two tubes. IPTG was added to both tubes at a final concentration of 0.4 μM, but MUG was added to only one of the two tubes at a final concentration of 100 μg/ml. The bacteria in each tube were cultured for 3 h before being observed under a long-wave UV lamp.

| Confocal microscopy
Fluorescence analysis was performed using a TCS SP5 confocal laser scanning microscope (Leica). GFP was excited at 488 nm and the emitted light captured at 500-550 nm, and mCherry was excited at 561 nm and emission light captured at 570-630 nm. For analysis of colocalization assays, multitracking was used to prevent emission cross-talk between the channels. Images were captured digitally and handled using Leica TCS software. Postacquisition image processing was done with Photoshop v. 7.0 software (Adobe Systems Inc.). Adams, UK for help in correcting the English in the manuscript.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data supporting the findings of this study are available from the corresponding author upon reasonable request.